2 MATERIALS AND METHODS
In 2021, a clinical (lung) sample,
TZJ2134, was collected in Shandong Province of China from a pig with
suspected PRRSV infection. Tissue sample processing, RNA extraction,
cDNA preparation, RT–PCR and genome sequencing were performed as
described previously (Leng et al., 2014; Zhang. et al., 2015). TZJ2134
was identified as PRRSV-1 by RT–PCR with the primer L12. Based on the
complete genomic sequence of PRRSV-1, eight primer pairs were designed
for RT–PCR amplification and sequencing (Table S2), and two overlapping
fragments of TZJ2134 were amplified by RT–PCR. Each PCR product was
purified with the E.Z.N.A.® Gel Extraction Kit - Omega Bio-Tek, cloned
into pMD18-T according to the manufacturer’s instructions, and then
submitted to Comate Bioscience Co., Ltd. (Changchun, China) for
sequencing.
Sequence analysis was performed with DNASTAR (version 7.1) software. The
resulting sequences were assembled using SeqMan. Phylogenetic trees and
molecular evolutionary analyses were constructed by using the
neighbor-joining method in MEGA 7.0. with 1000 bootstrap replicates for
each node (Kumar, Stecher, & Tamura, 2016). The generated phylogenetic
tree was annotated using iTOL online software (https://itol.embl.de/)
(Letunic & Bork, 2021). Sixty-five strains of PRRSV-1 available in
GenBank were used for comparative sequence analyses in this study.
To test for recombination, the
obtained sequence was screened using
recombination detection program 4 (RDP4) (Martin et al., 2015). Seven
different algorithms (RDP, GeneConv, BootScan, MaxChi, Chimera, SiScan,
and 3Seq) embedded in the RDP4 software package with Bonferroni
correction were utilized to detect recombination events and breakpoints.
Detection using four or more of the seven methods implemented in RDP4
was taken as significant evidence for recombination. Recombination
events were further confirmed by SimPlot 3.5.1 (Lole et al., 1999), and
boot scanning analysis was performed with a 200-bp window, sliding along
the genome alignment with a step size of 20 bp. The other three strains
(PRRS-FR-2014-56-11-1,
DK-2011-05-23-9 and OLot/91 strain) with high homology to TZJ2134-(A+B)
were analyzed by SimPlot 3.5.1 (Lole et al., 1999) using the same
method.